The intracellular sorting of pro-neuropeptides to the regulated secretory pathway (RSP) is essential for processing, storage and release of active hormones in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and proinsulin to the regulated secretory pathway (RSP) was investigated. We show that these pro-proteins undergo homotypic oligomerization, as a concentration step, as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network(TGN)where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a concensus sorting motif consisting of two acidic residues, 12-15A apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7A away from the acidic residues which are necessary for sorting to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC and pro-insulin was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, Arg255 and Lys260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP . We showed that CPE is a transmembrane protein which is anchored via its C-terminal amphipathic domain to unique cholesterol-glycosphingolipid rich microdomains known as rafts, in the TGN. Cholesterol depletion by treatment of cells with lovastatin or truncation of C-terminus of CPE resulted in lack of sorting of CPE and POMC to the RSP. Thus membrane association is essential for the prohormone sorting receptor function and the sorting of CPE at the TGN. Transfection of a mutant CPE with Arg255 and Lys260 mutated to alanine in a CPE null clone of Neuro2a cells, and transfection of a dominant negative CPE mutant into AtT-20 cells caused missorting of POMC to the constitutive pathway, indicating that the basic residues in the sorting domain of CPE interacts with the acidic residues in the POMC sorting signal in vivo to effect sorting to the RSP. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones to the regulated secretory pathway in neuro-endocrine cells. In addition, our studies also showed that high levels of secreted mutant proinsulins in the plasma of patients with hyperproinsulinemia are due to defects in sorting of the mutant proinsulins to the regulated secretory pathway, stemming from their inability to bind to the sorting receptor, CPE as a result of genetic alterations to their structure. Our studies also show that CPE is sorted by a mechanism involving association with lipid rafts. Furthermore, CPE, upon exocytosis is recycled back to the TGN by an ARF6 dependent-pathway and reused. In another project, we uncovered a master on/off switch , chromogranin A (CgA), that controls the formation of large dense-core granules(LDCG) at the TGN in neuroendocrine cells. In a mutant endocrine cell line, 6T3, lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. We compared gene expression in 6T3 cells lacking LDCGs and 6T3cells stably transfected with CgA using microarrays. Aquaporin 1(AQ1, a water channel) and the granuphilin genes were highly up-regulated in 6T3 cells expressing CgA, indicating a new role of CgA at the transcriptional level. These proteins appear to be in secretory granules and may play an important role in regulating exocytosis and hormone secretion in endocrine and neuroendocrine cells.